T7 RNA polymerase (E.C. 2.7.7.6.; herein also referred to as “T7 polymerase” or “T7”) is a monomeric bacteriophage encoded DNA directed RNA polymerase which catalyzes the formation of RNA in the 5′→3′ direction. In the process of initiation of transcription T7 recognizes a specific promoter sequence, the T7 promoter. T7 consists of 883 amino acids and has a molecular weight of 99 kDa. On the level of amino acid sequence T7 is highly homologous to T3 RNA polymerase and, to a lesser extent, SP6 RNA polymerase. The three-dimensional structure of T7 is very similar to other polymerases with different template and substrate specificities, despite low sequence similarity. T7 consists of different domains, the N-terminal domain, the “thumb”, the “palm” and the “fingers” (Sousa, R., and Mukherjee, S., Prog. Nucl. Acid Res. Mol. Biol. 73 (2003) 1-41).
Detailed studies of the transcription reaction showed that the enzyme acts like a molecular machine showing well concerted movements of flexible parts of the enzyme (Steitz, T. A., EMBO J. 25 (2006) 3458-3468; Steitz, T. A., Curr. Opin. Struct. Biol. 14 (2004) 4-9; Yin, Y. W., and Steitz, T. A., Cell 116 (2004) 393-404).
Several structures of T7 in complex with promoter DNA were solved and are available in the Protein Data Bank (pdb). The structure of the initiation complex of T7 RNA polymerase was solved at high resolution (Cheetham, G. M. T., et al., Nature 399 (1999) 80-83; Cheetham, G. M. T., and Steitz, T. A., Science 286 (1999) 2305-2309). The structure of the elongation complex solved at 2.9 A resolution showed the rearrangement of the N-terminal region (Tahirov, T. H., et al., Nature 420 (2002) 43-50). The structural studies showed that the conformation of the N-terminal domain changes between the initiation and elongation phases. Recently, the structure of transcribing T7 in transition from initiation to elongation phase was described (Durniak, K. J., et al., Science 322 (2008) 553-557).
The cloning and the expression of the gene encoding T7 has been described (Studier et al., U.S. Pat. No. 4,952,496). T7 has been studied intensively by mutagenesis to explore the conformational changes during transcription (Ma, K., et. al., Proc. Nat. Acad. Sci. 102 (2005) 17612-17617), to facilitate promoter clearance (Guillerez, J., et al., Proc. Natl. Acad. Sci. 102 (2005) 5958-5963) or to study the abortive cycling phenomenon (He, B., et al., J. Mol. Biol. 265 (1997) 275-288). Bonner, G., et al., J. Biol. Chem. 269 (1994) 25120-25128 described a set of active site mutants with altered elongation rates.
Due to the promoter specificity and high RNA polymerase enzymatic activity, T7 is useful for a variety of applications in molecular biology. In the field of recombinant protein expression T7 is used for the high-level expression of recombinant genes in E. coli (Studier, F. W., and Moffat, B. A., J. Mol. Biol. 189 (1986) 113-130). The synthesis of defined oligoribonucleotides was described by Milligan, J. F., et al., Nucl. Aids Res. 15 (1987) 8783-8798.
In addition, T7 is used in nucleic acid amplification methods for diagnostic purposes. A first example for such use is a technique known as “Nucleic Acid Sequence Based Amplification” (NASBA). This process comprises the steps of (a) adding a RNA template to a reaction mixture, wherein a first primer anneals to a complementary site at the 3′ end of the template; (b) reverse transcribing a DNA strand complementary to the RNA template, wherein a RNA/DNA heteroduplex is formed; (c) degrading the RNA strand of the heteroduplex by way of RNaseH activity; (d) annealing a second primer to the 5′ end of the DNA strand; (e) repeatedly synthesizing a complementary RNA strand with T7 RNA polymerase, wherein the synthesized RNA strand can serve again as a template in step (a). The NASBA technique has been used to develop rapid diagnostic tests for several pathogenic viruses, particularly those with single-stranded RNA genomes.
A further example for a diagnostic isothermal amplification method is “Transcription mediated amplification” (TMA) known to be one of the most sensitive detection assays for hepatitis C virus RNA in patient serum. For amplification of target RNA, two enzymes are used which are reverse transcriptase (RT) and T7 RNA polymerase. Complementary DNA (cDNA) of sample RNA is generated by RT with RNAse H activity and a primer containing a T7-promoter at the 5′-end. The RNA resulting of the RNA-DNA duplex is degradated by RNAse H activity of the RT. Another primer then binds to the cDNA already containing the T7-promoter sequence from the first primer and a double-stranded DNA is synthesized by the DNA polymerase activity of the RT. The T7 RNA polymerase recognizes the T7-promoter sequence within the double-stranded DNA molecule and synthesizes numerous RNA antisense transcripts. Each of the newly produced RNA amplicons re-enters the TMA process and serves as a template for a new round of RT to double-stranded DNA including the T7-promoter and transcription of antisense amplicons. The circulation of antisense transcripts into the amplification process results in exponential amplification of target RNA.
For NASBA, TMA and related methods as well as for other applications it would be advantageous if the reaction temperature could be elevated to improve the reaction kinetics. E.g., higher reaction temperatures of isothermal amplification could allow the amplification of RNA having secondary structures. It has also been shown with the polymerase chain reaction (PCR) technology that high annealing temperatures allow the specific hybridization of a primer to its target resulting in a highly specific amplification. With the same advantage, more thermostable enzymes could in principle also be applied isothermal amplifications.
Therefore, there is a need of a T7 RNA polymerase with improved stability and activity at higher reaction temperatures.
The stability of T7 RNA polymerase has been studied extensively. Thermal and urea-induced unfolding of was studied by Protasevich et al. using calorimetry, circular dichronism and fluorescence (Protasevich, I. I., et al., FEBS Lett 349 (1994) 429-432). Under the conditions used the enzyme unfolded at 48.3° C. Thermal unfolding was also studied by Griko et al. using calorimetric methods (Griko, Y., et al., Prot. Sci. (2001) 845-853). A smaller 22 kDa N-terminal part of the enzyme was shown to increase the thermostability of the C-terminal 77 kDa domain.
By introducing point mutations in the sequence of the wild-type enzyme T7 variants were generated in which the stability of T7 RNA polymerase was enhanced. The U.S. Pat. No. 6,524,828 and EP 1 261 696 describe four distinct amino acid exchanges in the T7 RNA polymerase polypeptide (Ser430Pro, Ser633Pro, Phe849Ile and Phe880Tyr) which stabilize the enzyme. Combinations of two or more of these mutations in a modified T7 polypeptide result in even more stable enzyme variants.
The aim of the present invention was to extend the collection of stabilizing mutations by creating novel mutations in T7 RNA polymerase which lead to improved stability. It is further desired to combine several of these mutations in a single T7 variant (double-, triple-, quadruple-, multiple-mutant), provided the combined mutations lead to an even increased stability, that is to say thermostability. According to the invention, new mutations are found giving rise to T7 variants which exhibit high stability in thermal unfolding assays.